Triple negative breast cancer poses a persistent clinical challenge given that it is refractory to most available endocrine or targeted therapies. Indeed, patient prognosis is dim in light of shorter median time to relapse and death compared to other breast cancer subtypes. Broadly speaking, our group is working to develop an improved therapy regimen for this patient population. We propose to achieve this by combining 2 separate treatment modalities [i.e. ceramide nanoliposomses (CNL) and image-guided focused ultrasound (FUS) ablation/heating] that we postulate will offer therapeutic synergy. Our expectation of synergy between CNL and FUS is based on evidence that (i) they are both capable of reducing, perhaps through distinct mechanisms, immunosuppression in the tumor microenvironment and (ii) the delivery of systemically administered liposomes is markedly enhanced in FUS-targeted tumors. The proposal consists of 2 specific aims. Aim 1 will be to determine which FUS regime (sub-ablative heating or partial thermal ablation) most effectively enhances the treatment efficacy of ceramide nanoliposomes against breast cancer. FUS will be applied to 4T1 (triple negative) breast tumors in combination with CNL administration. Determination of the most efficacious FUS regime will be made based upon tumor growth and animal survival data. Mass spectrometry of excised tumors will ascertain whether FUS enhances ceramide delivery. We will also perform comprehensive immunological analyses on tumor tissue, tumor draining lymph nodes (LNs), non-draining LN, spleen, and blood. Aim 2 will then be to investigate whether the most therapeutically efficacious combination of FUS and CNL is associated with toxicity. We will repeat the most efficacious FUS+CNL combination from Aim 1 and assess toxicity (liver and cardiac enzymes, cardio/neural and pulmonary physiological parameters, and hematological and immunological markers). Finally, we will perform ex-vivo studies to examine whether FUS application affects CNL chemistry. Going forward, note that Co-PI Kester has already successfully received an IND from the FDA (IND 109487) to initiate a phase 1 trial. If the proposed studies are successful, a follow-on phase 1/2 trial with CNL and FUS would ensue at our institution.